*Target (protein/gene name): New Delhi Metallo Beta-Lactamase *NCBI Gene # or RefSeq#:
NCBI Gene#: 14858116 *Protein ID (NP or XP #) or Wolbachia#: NC_021180.1 *Organism:
Klebsiella pneumoniae Etiologic Risk Group : RG1 *Background/Disease Information: For a century, modern society has relied on beta-lactam drugs such as penicillin-G to treat serious gram positive as well as gram negative bacterial infections. However, heavy reliance of these drugs has also selected for beta-lactamase synthesis as a form of an antimicrobial resistance mechanism in bacteria. Beta-lactamase cleaves the four-membered beta-lactam ring through hydrolysis, rendering it ineffective against infections. In conditions where antibiotic overuse is widespread, this spread of this mechanism via horizontal gene transfer has resulted in deadly versions of minor infections. A recently discovered beta-lactamase, New Delhi metallo beta-lactamase (NDM-1), resists most lactam-based inhibitors and is effective against a wide range of antibiotics of size and shape.
Essentiality of this protein: Essential in the presence of beta-lactam antibiotics such as ampicillin and penicillin to confer resistance Complex of proteins: There are identical proteins Chain A and B for 3Q6X. Druggable Target: Druggability index: 0.5 for Class A Beta Lactamase
*EC#:
3.5.2.6
Link to BRENDA EC# page:
Does not exist for NDM-1/K. pneumoniae
Following picture has correct reagent and product Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrometric or Titrimetric assay Reagents:
Thomas PW, Zheng M, Wu S, Guo H, Liu D, Xu D, Fast W. 2011: Assay solution: 0.1M Hepes, pH 7, 20uM ZnSO4 and 0.1mM substrate in water.
NDM-1 diluted in 50mM Tris-HCl, pH 7, and 150mM NaCl. Substrate: Nitrocefin, Calbiochem, 5mg: $109.00
100 mM Tris HCl Buffer with 0.1% (w/v) Bovine Serum Albumin, pH 7.0 at 25°CAmpicillin/beta-lactam Solution (Control)
10 mM Sodium Hydroxide Solution Standardized (NaOH)-- link to Sigma (or other company) page for assay or assay reagents
Structure Available (PDB or Homology model)
-- PDB # :3Q6X
Current Inhibitors: Aztreonam and possibly Clavulanic Acid Expression Information (has it been expressed in bacterial cells): Purification Method: His-6, Nickel or Strep tag
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): From Fast document* MELPNIMHPVAKLSTALAAALMLSGCMPGEIRPTIGQQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGF GAVASNGLIVRDGGRVLVVDTAWTDDQTAQILNWIKQEINLPVALAVVTHAHQDKMGGMDALHAAGIATY ANALSNQLAPQEGMVAAQHSLTFAANGWVEPATAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGGCLI KDSKAKSLGNLGDADTEHYAASARAFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR
*length of your protein in Amino Acids: 270 Amino Acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28.6107 kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
e280 = 28,500 M−1 cm−1 (from Kim paper)
*GC% Content for gene:
60.820045558087% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Buffers fromShen 2013
Harvested cells by centrifugation at 10000xg for 15 min at 4 C, suspended in 30 mM phosphate-buffered saline, pH 7.3
Lysis: 30 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.3
Wash: 30 mM Tris-HCl, 0.5 M NaCl, 40 mM imidazole, pH 7.3
Elution: 30 mM Tris-HCl, 0.5 M NaCl, 100 mM imidazole, pH 7.3
Did Ni-NTA twice, then dialyzed against 2 L of 20 mM HEPES, 100 uM Zn(NO3)2, pH 6.8 overnight at 4 C.
*NCBI Gene # or RefSeq#:
NCBI Gene#: 14858116
*Protein ID (NP or XP #) or Wolbachia#:
NC_021180.1
*Organism:
Klebsiella pneumoniae
Etiologic Risk Group : RG1
*Background/Disease Information:
For a century, modern society has relied on beta-lactam drugs such as penicillin-G to treat serious gram positive as well as gram negative bacterial infections. However, heavy reliance of these drugs has also selected for beta-lactamase synthesis as a form of an antimicrobial resistance mechanism in bacteria. Beta-lactamase cleaves the four-membered beta-lactam ring through hydrolysis, rendering it ineffective against infections. In conditions where antibiotic overuse is widespread, this spread of this mechanism via horizontal gene transfer has resulted in deadly versions of minor infections. A recently discovered beta-lactamase, New Delhi metallo beta-lactamase (NDM-1), resists most lactam-based inhibitors and is effective against a wide range of antibiotics of size and shape.
Essentiality of this protein: Essential in the presence of beta-lactam antibiotics such as ampicillin and penicillin to confer resistance
Complex of proteins: There are identical proteins Chain A and B for 3Q6X.
Druggable Target: Druggability index: 0.5 for Class A Beta Lactamase
*EC#:
Does not exist for NDM-1/K. pneumoniae
Following picture has correct reagent and product
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrometric or Titrimetric assay
Reagents:
Thomas PW, Zheng M, Wu S, Guo H, Liu D, Xu D, Fast W. 2011:
Assay solution: 0.1M Hepes, pH 7, 20uM ZnSO4 and 0.1mM substrate in water.
NDM-1 diluted in 50mM Tris-HCl, pH 7, and 150mM NaCl.
Substrate: Nitrocefin, Calbiochem, 5mg: $109.00
100 mM Tris HCl Buffer with 0.1% (w/v) Bovine Serum Albumin, pH 7.0 at 25°CAmpicillin/beta-lactam Solution (Control)
10 mM Sodium Hydroxide Solution
Standardized (NaOH)-- link to Sigma (or other company) page for assay or assay reagents
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/penicillbenzyl.pdf
-- link (or citation) to paper that contains assay information:
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024621
-- List cost and quantity of substrate reagents and supplier
Supplier Sigma Aldrich
Ampicillin
157.50
Santa Cruz Biotechnology, inc
Bovine Serum Albumin-100g
$136
Structure Available (PDB or Homology model)
-- PDB # :3Q6X
Current Inhibitors: Aztreonam and possibly Clavulanic Acid
Expression Information (has it been expressed in bacterial cells):
Purification Method: His-6, Nickel or Strep tag
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): From Fast document*
MELPNIMHPVAKLSTALAAALMLSGCMPGEIRPTIGQQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGF
GAVASNGLIVRDGGRVLVVDTAWTDDQTAQILNWIKQEINLPVALAVVTHAHQDKMGGMDALHAAGIATY
ANALSNQLAPQEGMVAAQHSLTFAANGWVEPATAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGGCLI
KDSKAKSLGNLGDADTEHYAASARAFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR
*length of your protein in Amino Acids: 270 Amino Acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28.6107 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
e280 = 28,500 M−1 cm−1 (from Kim paper)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atggaattgcccaatattatgcacccggtcgcgaagctgagcaccgcatta 61
62 gccgctgcattgatgctgagcgggtgcatgcccggtgaaatccgcccgacgattggccag 121
122 caaatggaaactggcgaccaacggtttggcgatctggttttccgccagctcgcaccgaat 181
182 gtctggcagcacacttcctatctcgacatgccgggtttcggggcagtcgcttccaacggt 241
242 ttgatcgtcagggatggcggccgcgtgctggtggtcgataccgcctggaccgatgaccag 301
302 accgcccagatcctcaactggatcaagcaggagatcaacctgccggtcgcgctggcggtg 361
362 gtgactcacgcgcatcaggacaagatgggcggtatggacgcgctgcatgcggcggggatt 421
422 gcgacttatgccaatgcgttgtcgaaccagcttgccccgcaagaggggatggttgcggcg 481
482 caacacagcctgactttcgccgccaatggctgggtcgaaccagcaaccgcgcccaacttt 541
542 ggcccgctcaaggtattttaccccggccccggccacaccagtgacaatatcaccgttggg 601
602 atcgacggcaccgacatcgcttttggtggctgcctgatcaaggacagcaaggccaagtcg 661
662 ctcggcaatctcggtgatgccgacactgagcactacgccgcgtcagcgcgcgcgtttggt 721
722 gcggcgttccccaaggccagcatgatcgtgatgagccattccgcccccgatagccgcgcc 781
782 gcaatcactcatacggcccgcatggccgacaagctgcgctga
*GC% Content for gene:
60.820045558087%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Buffers from Shen 2013
Harvested cells by centrifugation at 10000xg for 15 min at 4 C, suspended in 30 mM phosphate-buffered saline, pH 7.3
Lysis: 30 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.3
Wash: 30 mM Tris-HCl, 0.5 M NaCl, 40 mM imidazole, pH 7.3
Elution: 30 mM Tris-HCl, 0.5 M NaCl, 100 mM imidazole, pH 7.3
Did Ni-NTA twice, then dialyzed against 2 L of 20 mM HEPES, 100 uM Zn(NO3)2, pH 6.8 overnight at 4 C.